Phosphorylation is a ubiquitous protein post-translational modification that plays a critical role in the cell signaling pathways governing cellular functions. The dynamic activities of kinase and phosphatase enzymes ensure proper phosphorylation in healthy cells. However, when phosphorylation becomes unregulated, generally through aberrant kinase or phosphatase activities, cells become diseased. In fact, various drugs targeting kinases and phosphatases have been developed to treat diseases from cancers to Alzheimer's. As a result, the monitoring of kinases and phosphatases, and the signaling pathways that they control, represents an important goal towards characterizing disease states and developing new drugs. Unfortunately, the available methods to monitor cellular phosphorylation and map the complex signaling pathways controlled by phosphorylation are challenging. New methods are necessary to fully characterize phosphorylation, cellular signaling, and disease formation. The long-term goal of this project is to rigorously characterize kinase and phosphatase activities in cells to map signaling pathways in healthy and diseased states. The scientific foundation established in the prior funding period is our revelation that kinases promiscuously accept ?-phosphoryl modified ATP analogs as cosubstrates. Based on this cosubstrate promiscuity, we pioneered several kinase-catalyzed labeling methods. We aim here to apply kinase-catalyzed labeling to several critical challenges in cell signaling research. First, we will develop kinase-catalyzed biotinylation to distinguish dynamic versus nondynamic phosphorylation events (Specific Aim 1). Second, kinase-catalyzed crosslinking will be established to monitor transient phosphorylation-mediated protein-protein interactions that are challenging to characterize by traditional methods, focusing on kinase-substrate pairs (Specific Aim 2). Phosphorylation-dependent crosslinking has the exciting potential to provide network-level details about signal transduction pathways, which is a critical yet underdeveloped need in cell signaling studies. A bold new direction for the project is application of kinase-catalyzed labeling to Ser/Thr phosphatase substrate identification (Specific Aim 3). Few strategies are available to characterize substrates of Ser/Thr phosphatases, making our innovative approach crucial to embolden cell signaling research. By studying both kinases and phosphatases, kinase-catalyzed labeling experiments will build a more detailed and complete picture of the complex cell signaling pathways governing disease. In total, the chemical tools established in this project will provide innovative and unprecedented approaches to monitor the role of phosphorylation in biology.